Fibronectin is useful in therapeutic treatment because of the role that it plays in cellular adhesion, blood coagulation, malignant transformation, reticuloendothelial system function, and embryonic differentiation. Fibronectin's role in cellular adhesion and in promoting epithelial cell outgrowth makes it desirable for treatment of ophthalmic wounds, particularly corneal disorders. Other growth factors have also been identified as being useful as healing accelerators for treatment of ophthalmic wounds. For example, recombinant human epithelial growth factor has been shown to accelerate corneal re-epithelialization following abrasive injury or alkali burn injury (Stern et al., "The Effects of Human Recombinant Epidermal Growth Factor on Epihelial Wound Healing", in Healing Processes in the Cornea, at 69-75 (C. E. Crosson and H. E. Kaufman, eds.) (1989). Similarly, fibroblast growth factor has also been reported to stimulate corneal healing (Countois, Y. et al., 181 C. R. Soc. Biolo, 491 (1987). Numerous other growth promoting substances have also been identified (e.g., interleukin 6, platelet-derived growth factor, etc.) and may be useful in accelerating ophthalmic wound healing. Ophthalmic wounds can be caused in many ways, for example, by puncture, physical trauma, acid splash, surgical incisions, chemical burns, or lacerations. It is believed that fibronectin promotes the migration of epithelial cells over the wound surface and promotes binding of the epithelial cells to the wound surface to provide a permanent closure of the wound. This process may stimulate the production of endogenous growth factors, such as fibroblast growth factors.
To treat an ophthalmic wound with fibronectin, the fibronectin should be applied by means of an ophthalmic solution. Multi-dose ophthalmic solutions to be used by a single user are the typical mode of applying ophthalmic solutions. One problem in using fibronectin arises from U.S. Federal Food and Drug Agency ("FDA") regulations which require the addition of a preservative to inhibit bacterial growth in a multi-dose ophthalmic solution.
Benzalkonium chloride is the most commonly used preservative in ophthalmic solutions, but it cannot be used with fibronectin because it inhibits the wound healing activity of the fibronectin. Chlorobutanol and phenylethyl alcohol are accepted alternative preservatives in ophthalmic solutions, but they also cannot be used with fibronectin. Chlorobutanol is hydrolyzed in a neutral pH solution. Phenylethyl alcohol cannot be used because it inhibits fibronectin's wound healing activity. Similarly, preservatives made from sodium dehydroacetate or cetylpyridinium dichloride inhibit the wound healing activity of fibronectin. Thimerosal does not inhibit fibronectin's wound healing activity but thimerosal's mercury content and the toxicity problems associated with mercury make it unsuitable for use as a preservative in an ophthalmic solution.
A second difficulty in using fibronectin in ophthalmic settings are problems related to the poor solubility and stability of fibronectin in an aqueous solution. Because of fibronectin's poor storage stability in solution it is a standard practice to lyophilize a solution of fibronectin with a stabilizing agent, usually a neutral amino acid, monosaccharide, disaccharide, or sugar alcohol. A solvent is added to the lyophilized fibronectin just before use. The disadvantage of this method is that the dissolving of the lyophilized preparation in the solvent, typically water, takes a long time and the resulting solution is often turbid because of fibrous insoluble matter.
One method to address this lyophilization problem has been disclosed in Ohmura U.S. Pat. No. 4,565,651. In the Ohmura patent, prior to lyophilization, both albumin and at least one stabilizer selected from neutral amino acids, monosaccharides, disaccharides, and sugar alcohols are added to a fibronectin-containing aqueous solution which is then lyophilized. According to Ohmura, when his lyophilized fibronectin is dissolved in water, the dissolution time is rapid, with little or no turbidity. For an ophthalmic solution, however, the lyophilized fibronectin of Ohmura may prove unacceptable because of the presence of an additional protein, albumin. Albumin renders preservatives less effective and may also interfere with the function of fibronectin. Additionally, the lyophilized fibronectin produced by the method of the Ohmura patent tends to cake up and then does not dissolve easily.